The invention involves viral vectors that can be used to transduce a target cell, i.e., to introduce genetic material into the cell. The targets of interest are eukaryotic cells and particularly human cells. The transduction can be done in vivo or in vitro. More particularly the invention concerns viral vectors, that can be used to transduce one from among many types of cell that has a particular acceptor molecule exposed on the target cell""s surface.
A variety of viral based vectors have been employed to transfer and to express a gene of interest into a eukaryotic target cell. Recombinant DNA techniques are used to replace one or more of the genes of the virus with the gene of interest operably linked to a promoter that is functional in the target cell. The construct, termed a viral vector, infects the target cell, using the physiological infective xe2x80x9cmachineryxe2x80x9d of the virus, and expresses the gene of interest instead of the viral genes. Because not all the genes of the virus are present in the vector, infection of the target by the vector does not produce viral particles. Viruses that have been used to infect human or mammalian target cells include herpes virus, adenovirus, adeno associated virus and derivatives of leukemia-type retroviruses. Among the retroviruses of particular interest in the transduction of cells of human origin are constructs based on amphotropic retroviruses.
Retroviruses are particularly well suited for transduction of eukaryotic cells. The advantages of a vector based this type of virus include its integration into the genome of the target cell so that the progeny of the transduced cell express the gene of interest. Secondly, there are well developed techniques to produce a stock of infectious vector particles that do not cause the production of viral particles in the transduced target cell. Lastly, the production and purification of stocks vector particles having titers of 106 TCIU/ml can be accomplished.
One disadvantage of the use of retroviral vectors is that there is presently no practical general, method whereby a particular tissue or cell type of interest can be specifically transduced. Previous efforts to this end have included surgical procedures to limit to specific organs the physical distribution of the viral vector particles. Ferry, N., et al., 1991, Proc.Natl.Acad.Sci. 88:8377.
Alternatively, practitioners have taken advantage of the fact that type C retroviruses only infect dividing cells. Thus, a population of cells, e.g., bone marrow cells, was removed from a subject and cultured ex vivo in the presence of growth factors specific for the specific target cell which, thus, comprises most of dividing cells in the culture. See, e.g., Wilson, J. M., et al., 1990, Proc.Natl.Acad.Sci. 87:439-47; Ohashi, T., et al., 1992, Proc.Natl.Acad.Sci. 89:11332-36. After transduction the dividing cells must be harvested and, for many purposes, reimplanted into the subject. The technical difficulties of the ex vivo culture technique combined with the unavailability of growth factors of specific for some types of cells have limited the application of this approach.
A second difficulty presented by the use retroviral based vectors is that a retroviral particle contains two copies of its genome. There is a nonzero possibility of a genetic recombination between the alleles of the viral particles. Such recombination can give rise to a replication competent virus that can cause the production of infectious particles by the target cell. In contrast to herpes virus or adenovirus infection retroviral infections are not necessarily self-limiting.
Notwithstanding these difficulties retrovirus vectors, based on amphotropic murine leukemia retroviruses that infect human cells, have been approved for use in human gene therapy of certain diseases, for example adenosine deaminase and low density lipoprotein receptor deficiencies and Gaucher""s Disease. See, e.g., Miller A. D., 1992, Nature 357:455; Anderson, W. F., 1992, Science 256:808.
One approach to overcoming the limitations of using amphotropic retrovirus vectors in human cells has been to mutate the gene encoding the protein on the viral surface that determines the specificity of infection of the virus, the gp70 protein. Using recombinant DNA technology a xe2x80x9cmutantxe2x80x9d virus is constructed that has had small regions of the gp70 sequence replaced by predetermined sequences. The limits of this approach are set by the requirement for knowledge of the sequence that will enable infection of the target of interest. However, when this knowledge was available, the anticipated alteration in viral specificity has been observed. Valsesia-Wittmann, S., 1994, J.Virol. 68:4609-19.
An alternative to altering the specificity of binding of the gp70 protein itself is to employ a second, novel structure that binds or is bonded to both the viral particle and to the target cell. Such novel, independently functioning molecules can be thought of as molecular adapters which, together with the viral particle form a vector complex. In one example of this approach, lactose molecules were covalently coupled, by a non-specific reaction, to the envelope proteins of an ecotropic retrovirus, which does not normally infect human cells. A human hepatocellular carcinoma that was known to have receptors for lactose-containing proteins was found to be susceptible to transduction by this vector complex, although the integration of the transduced gene of interest in the target cell chromosome was not directly demonstrated. Neda, H., et al., 1991, J.Biol.Chem. 266:14143. No evidence of expression was observed in a hepatocellular carcinoma that lacked the lactose specific receptor. The method of Neda results in a variable number of binding sites for the exposed acceptor on the target cell, attached to each derivatized or bound envelope protein and, of course, is limited to the case wherein the target cell has a lactose receptor.
Another approach to the use of adapter molecules involved an adapter that was not covalently coupled to the vector. The use of this type of adapter has been attempted by Roux and his colleagues, who have published several reports that relate to this strategy. Patent Publication FR 2,649,119 to Piecheczyk, Jan. 4, 1991; Roux P., et al., 1989, Proc. Natl. Acad. Sci. 86:9079-83; Etienne-Julan, M., et al., 1992, J. Gen. Virol. 73:3251-55. Roux and colleagues have constructed adapters from two types of proteins, both typically antibodies, by biotinylating the proteins and utilizing avidin or streptavidin tetramer, a protein which binds four biotin molecules, to form aggregates of up to four of the biotinylated proteins. The first type of proteins was an anti-gp70 antibody, which binds to the viral particle. The second type of protein was one of a variety of proteins that specifically bind to the target cells and could be an antibody or other protein. The adapter of Roux contained a streptavidin tetramer and four other protein molecules. Each adapter contained one streptavidin tetramer, but the aggregates were otherwise random, i.e., all possible combinations of the two other types of proteins were possible including aggregates having only anti-gp70 and only the target cell specific protein.
To offset the difficulties attendant with the use of mixtures of randomly aggregated proteins, Roux did not employ pre-formed adapters but rather constructed the adapters, in situ, i.e., on the surface of the target cell, by successively exposing the cells to the target cell specific protein, the streptavidin, the anti-gp70 and lastly to the viral vector itself. Even when constructed in situ, the adapter molecules of Roux consisted of a random mixture having no predetermined number of viral or target cell binding sites.
To allow for the completion of this multistep process the target cells must be prevented, by some means, from internalizing the components of the aggregates prior to their completion. The method adopted by Roux was to reduce the temperature of the culture. Thus, the work of Roux does not yield a system that can be used at all in an in vivo setting. Even ex vivo, the complex of adapter and vector must be constructed in a multistep process during which the metabolism of the target cell must be inhibited.
The invention concerns the use of viral vectors that have proteins on the surface of the viral particles, hereinafter termed envelope proteins, that do not bind to the target cell of interest. The invention involves a new type of adapter that provides a fixed, predefined valence, i.e., number of binding sites, specific for an exposed acceptor molecule on the target cell, on each envelope protein molecule having such an adapter. Because the binding sites for the viral particle and the target cell are different, the adapters of the invention can be constructed in the absence of the target cell. The preformed complexes of adapters and viral particles that can then be used to transduce a gene of interest into the target cell.
In one embodiment, the invention comprises a complex of a viral vector and a non-covalently bonded difunctional molecule, i.e, a molecule having at least one site for linking with the virus and a predefined number of binding sites specific for the target cell of interest. In a particular embodiment of this type, the difunctional molecule has a single binding site for the virus and a single site for the target cell.
The invention also encompasses complexes comprising a viral vector in which some or all of the envelope protein molecules of the virus are modified by formation a new covalent bond. There are three different embodiments of this form of the invention.
Firstly, the adapter molecule can be a binding polypeptide that replaces at least about 25 amino acids of the envelope protein of the virus and which, thereby, forms a fusion protein. Such fusion proteins can be made by linking, through recombinant DNA techniques, the fragment of the gene encoding the fragment of the envelope protein to a fragment of a gene that encodes a parent protein having the same, desired binding specificity as the binding polypeptide. As used herein, a fusion protein is a protein having at least two blocks of contiguous sequence, of about 10 or more amino acids in length, that are derived from two different parent proteins. In a preferred embodiment, the viral particle contains a mixture of normal envelope proteins and fusion proteins.
Secondly the adapter can consist of a linking molecule and a polypeptide of an envelope protein/polypeptide fusion protein, as described above. The non-envelope polypeptide of the fusion protein is complementary to the linking molecule and is, hence, termed a linking polypeptide. In this embodiment, the linking molecule is not covalently bonded to the vector. Rather, the linking molecule is itself difunctional. It contains a ligand functionality, which is complementary to the linking polypeptide, affixed to the viral surface, and an acceptor binding portion which contains a predefined number of binding sites for an exposed acceptor on the target cell surface.
The third embodiment employs the linking molecule, as described above, but does not utilize an envelope protein/linking polypeptide fusion protein. Rather in this embodiment a linking site is covalently affixed to the envelope protein after the vector particle has been constructed.
The invention further provides methods whereby a one skilled in the art can determine whether a particular antibody is suitable for use as a target cell specific protein and encompasses methods of use of the preformed viral vector complexes.